Enhancement of microsphere specificity to purify human serum albumin from blood plasma.

Efforts to rapidly and easily supply purified human serum albumin (HSA) in remote areas during a pandemic are challenging. Here, we developed an HSA-imprinted microsphere (HSAIM) as a matrix to purify HSA from blood plasma on a small scale. HSAIMs were generated by encapsulating silica-3-(2-imidazoline-1-yl) propyltriethoxysilane-Cu2+-HSA (SiO2-IMEO-Cu2+-HSA) into agarose gel, and the stereospecific template for HSA was obtained by eluting the agarose gel. The physicochemical properties and performance of HSAIM were evaluated, and HSAIMs were applied to purify HSA from human blood plasma. Spherical HSAIMs had an average diameter of 51.2 ± 6.1 µm. HSAIMs had a maximum adsorption of 8.77 × 10-2 µmol HSA g-1 with an imprinting factor of 2.83, and the selectivity factors of BSA, thrombin and IgG were 0.96, 0.59 and 0.26, respectively. HSAIMs had a dynamic binding capacity (DBC10%) of 5.94 × 10-2 µmol HSA g-1 and could be reused up to 10 cycles with an ultimate recovery of 55.92%. HSA adsorption kinetics of HSAIM fitted to a pseudo-second-order mechanism, and HSA binding characteristics fit with a Sips isotherm model. For practical purposes, an initial blood plasma sample containing 24.9 ± 2.5 mg protein was pretreated with ethanol yielding 14.5 ± 4.6 mg protein, and further purification with HSAIM yielded 3.6 ± 1.1 mg protein. Starting with a blood plasma sample containing 149 type proteins, a single protein identified as HSA was obtained after final purification step with the HSAIM column, indicating that HSAIMs stereospecifically bound HSA. Hence, HSAIM was promising for blood plasma purification on a small scale.

Structural Characterization of Polycrystalline Titania Nanoparticles on C. striata Biosilica for Photocatalytic POME Degradation.

The biosilica shell of marine diatoms has emerged as a unique matrix for photocatalysis, owing to its sophisticated architecture with hierarchical nanopores and large surface area. Although the deposition of titania nanoparticles on diatom biosilica has been demonstrated previously, their photocatalytic activity has been tested only for degradation of pure compounds, such as dyes, nitrogen oxide, and aldehydes. The efficiency of such photocatalysts for degradation of mixtures, for instance, industrial wastewaters, is yet to be investigated. Furthermore, reports on the lattice structures and orientation of nanotitania crystals on biosilica are considerably limited, especially for the underexplored tropical marine diatoms. Here, we report an extensive characterization of titania-loaded biosilica from the tropical Cyclotella striata diatom, starting from freshly grown cell cultures to photodegradation of wastewaters, namely, the palm oil mill effluent (POME). As Indonesia is the largest palm oil producer in the world, photocatalytic technology could serve as a sustainable alternative for local treatment of POME. In this study, we achieved a 54% loading of titania on C. striata TBI strain biosilica, as corroborated by XRF analyses, which was considerably high compared to previous studies. Through visualization using HR-TEM, supported by SAED and XRD analyses, nanocrystal TiO2 appeared to be trapped in an anatase phase with polycrystalline characteristics and distinct crystallographic orientations. Importantly, the presence of C. striata biosilica lowered the band gap of titania from 3.41 eV to around 3.2 eV upon deposition, enabling photodegradation of POME using a broad-range xenon lamp as the light source, mimicking the sunlight. Kinetic analyses revealed that POME degradation using the photocatalysts followed quasi-first-order kinetics, in which the highest titania content resulted in the highest photocatalytic activity (i.e., up to 47% decrease in chemical oxygen demand) and exhibited good photostability throughout the reaction cycles. Unraveling the structure and photoactivity of titania-biosilica catalysts allows transforming marine diatoms into functional materials for wastewater photodegradation.

Enhanced Oil Production by the Tropical Marine Diatom Thalassiosira Sp. Cultivated in Outdoor Photobioreactors.

Microalgae-derived oils have potential as a biofuel feedstock. To produce microalgal oils at a large scale, large amounts of nutrients and energy are needed to grow the algae. In this study, we evaluated three types of agricultural fertilizer (AF)-based culture media (AF1, AF2, and AF3) based on a previously published enriched seawater (ES) medium to produce biomass and oils from Thalassiosira sp. Under laboratory conditions, the highest cell productivity of Thalassiosira sp. was obtained with the AF3 medium. Thalassiosira sp. cultured in the AF3 medium produced 10.4 ± 0.9 mg L-1 day-1oils, which is significantly higher than the 5.8 ± 0.7 mg L-1 day-1produced in the ES medium. The higher production was due to the presence of nitrate and trace elements, both of which played roles in enhancing biomass and oil content, respectively. During cell growth, resting spores appeared inside the cells and were a marker to harvest the cells. Because of the abundant availability of sunlight in the tropics during the year, the oil production of Thalassiosira sp. in the AF3 medium was scaled up using outdoor photobioreactors under different weather conditions (rainy and dry seasons). Thalassiosira sp. produced more unsaturated fatty acids during the rainy season and produced more saturated fatty acids during the dry season. This study also demonstrated that it was possible to culture Thalassiosira sp. under outdoor conditions using a low-cost agricultural fertilizer-based culture medium (AF3 medium) to produce biodiesel feedstock with an annual production of 8.1 ± 0.4 t ha-1 during the dry season and of 23.9 ± 6.8 t ha-1 during the rainy season.

A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad.

α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (ß/α)8-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia. In BmaN1 the third residue, the aspartic acid that acts as the transition state stabilizer, was replaced by a histidine. Three-dimensional structure modeling of the BmaN1 amino acid sequence confirmed the aberrant catalytic triad. Glucose and maltose were found as products of the action of the novel α-amylase on soluble starch, demonstrating that it is active in spite of the peculiar catalytic triad. This novel BmaN1 α-amylase is part of a group of α-amylases that all have this atypical catalytic triad, consisting of aspartic acid, glutamic acid and histidine. Phylogenetic analysis showed that this group of α-amylases comprises a new subfamily of the glycoside hydrolase family 13.

Effect of introducing a disulphide bond between the A and C domains on the activity and stability of Saccharomycopsis fibuligera R64 α-amylase.

Native enzyme and a mutant containing an extra disulphide bridge of recombinant Saccharomycopsis fibuligera R64 α-amylase, designated as Sfamy01 and Sfamy02, respectively, have successfully been overexpressed in the yeast Pichia pastoris KM71H. The purified α-amylase variants demonstrated starch hydrolysis resulting in a mixture of maltose, maltotriose, and glucose, similar to the wild type enzyme. Introduction of the disulphide bridge shifted the melting temperature (TM) from 54.5 to 56 °C and nearly tripled the enzyme half-life time at 65 °C. The two variants have similar kcat/KM values. Similarly, inhibition by acarbose was only slightly affected, with the IC50 of Sfamy02 for acarbose being 40 ± 3.4 µM, while that of Sfamy01 was 31 ± 3.9 µM. On the other hand, the IC50 of Sfamy02 for EDTA was 0.45 mM, nearly two times lower than that of Sfamy01 at 0.77 mM. These results show that the introduction of a disulphide bridge had little effect on the enzyme activity, but made the enzyme more susceptible to calcium ion extraction. Altogether, the new disulphide bridge improved the enzyme stability without affecting its activity, although minor changes in the active site environment cannot be excluded.

Construction of individual, fused, and co-expressed proteins of endoglucanase and ß-glucosidase for hydrolyzing sugarcane bagasse.

At least a combination of endoglucanase (EglII) and ß-glucosidase (BglZ) is required for hydrolyzing crystalline cellulose. To understand the catalytic efficiency of combination enzymes for converting biomass to sugars, EglII and BglZ were constructed in the form of individual, fused as well as co-expression proteins, and their activities for hydrolyzing sugarcane bagasse were evaluated. The genes, eglII isolated from Bacillus amyloliquefaciens PSM3.1 earlier and bglZ from B. amyloliquefaciens ABBD, were expressed extracellularly in Bacillus megaterium MS941. EglII exhibited both exoglucanase and endoglucanase activities, and BglZ belonging to the glycoside hydrolase 1 family (GH 1) showed ß-glucosidase activity. A combination of EglII and BglZ showed activity on substrates Avicel, CMC and sugarcane bagasse. Specifically for hydrolyzing sugarcane bagasse, fused protein (fus-EglII+BglZ), co-expression protein (coex-BglZ+EglII), and mixed-individual protein (mix-EglII+BglZ) produced cellobiose as the main product, along with a small amount of glucose. The amount of reducing sugars released from the hydrolyzing bleached sugarcane bagasse (BSB) using fus-EglII+BglZ and mix-EglII+BglZ was 2.7- and 4.2-fold higher, respectively, than steamed sugarcane bagasse (SSB), indicating the synergetic enzymes worked better on treated sugarcane bagasse. Compared with fus-EglII+BglZ and mix-EglII+BglZ, coex-BglZ+EglII released more mol reducing sugars from SSB, indicating the enzymes were potential for biomass conversion. Additionally, coex-BglZ+EglII acted on BSB 2.5-fold faster than fus-EglII+BglZ. Thus, coex-bglZ+eglII expression system was the best choice to produce enzymes for hydrolyzing sugarcane baggase.

ß-D-xylosidase from Geobacillus thermoleovorans IT-08: biochemical characterization and bioinformatics of the enzyme.

The gene encoding a thermostable ß-D-xylosidase (GbtXyl43B) from Geobacillus thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli; additionally, characterization and kinetic analysis of GbtXyl43B were carried out. The gene product was purified to apparent homogeneity showing M r of 72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum temperature and pH of 60 °C and 6.0, respectively. In terms of stability, GbtXyl43B was stable at 60 °C at pH 6.0 for 1 h as well as at pH 6-8 at 4 °C for 24 h. The enzyme had a catalytic efficiency (k cat/K M) of 0.0048 ± 0.0010 s(-1) mM(-1) on p-nitrophenyl-ß-D-xylopyranoside substrate. Thin layer chromatography product analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble xylan was eightfold higher than on soluble xylan. Bioinformatics analysis showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel ß-strands) and catalytic module (residues 157 to 604 forming five-bladed ß-propeller fold with predicted catalytic residues to be Asp287 and Glu476). CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is proposed as a novel CBM36 subfamily.

Screening, gene sequencing and characterising of lipase for methanolysis of crude palm oil.

Staphylococcus sp. WL1 lipase (LipFWS) was investigated for methanolysis of crude palm oil (CPO) at moderate temperatures. Experiments were conducted in the following order: searching for the suitable bacterium for producing lipase from activated sludge, sequencing lipase gene, identifying lipase activity, then synthesising CPO biodiesel using the enzyme. From bacterial screening, one isolated specimen which consistently showed the highest extracellular lipase activity was identified as Staphylococcus sp. WL1 possessing lipFWS (lipase gene of 2,244 bp). The LipFWS deduced was a protein of 747 amino acid residues containing an α/ß hydrolase core domain with predicted triad catalytic residues to be Ser474, His704 and Asp665. Optimal conditions for the LipFWS activity were found to be at 55 °C and pH 7.0 (in phosphate buffer but not in Tris buffer). The lipase had a K(M) of 0.75 mM and a V(max) of 0.33 mMmin(-1) on p-nitrophenyl palmitate substrate. The lyophilised crude LipFWS performed as good as the commonly used catalyst potassium hydroxide for methanolysis of CPO. ESI-IT-MS spectra indicated that the CPO was converted into biodiesel, suggesting that free LipFWS is a worthy alternative for CPO biodiesel synthesis.

Oil from the tropical marine benthic-diatom Navicula sp.

The potential of the tropical marine benthic-diatom Navicula sp. for biodiesel feedstock was investigated. Growth profiles were analyzed by changing nutrient compositions in three different media (Walne, plain seawater, and modified seawater) and irradiance intensities. Navicula sp. cells showed significant growth in Walne and modified seawater medium but not in plain seawater medium. The microalgae grew well in a pH range of 7.8-8.4, and the cells were very sensitive to the intensity of direct sunlight exposure. The average cell concentration obtained from the cultures in plain seawater, Walne, and modified seawater media at the beginning of the stationary phase was 0.70, 2.17, and 2.54 g/L, respectively. Electron spray ionization-ion trap-mass spectrometry showed that the triacylglycerols of the algae oil were identified as POP (palmitic-oleic-palmitic), POO (palmitic-oleic-oleic), and OOLn (oleic-oleic-linoleic). The oil productivity of Navicula sp. cultivated in Walne and modified seawater media was 90 and 124 µL L(-1) culture d(-1). The Navicula sp. biodiesel exhibited a kinematic viscosity of 1.299 mm(2)/s, density of 0.8347 g/mL, and internal energy of 0.90 kJ/mL.

Oil productivity of the tropical marine diatom Thalassiosira sp.

To understand the potential of cultivating tropical marine diatom Thalassiosira sp. to produce biofuel, biodiesel product properties and growth characteristics of Thalassiosira sp. in three different media were investigated. After medium evaluation, significant Thalassiosira sp. cell growth was observed in both Walne and enriched seawater media, but not in plain seawater medium. The microalgae grew well in alkaline condition (pH range of 8.0-8.8). The average biomass density cultured in Walne and enriched seawater media on the 6th day was 4.36 and 2.50 g L(-1), respectively. Based on ESI-IT-MS spectra, the TAGs of algal oil were identified as POP, POO, and SOO, and the FAMEs as oleic acid methyl ester. The oil productivity of Thalassiosira sp. cultured in Walne and enriched seawater media were 150 and 290 µL L(-1) d(-1), respectively. The density and kinematic viscosity of Thalassiosira sp. biodiesel were 0.857 g mL(-1) and 1.151 mm(2) s(-1).